The present invention relates to a method of estimating the fragmentation pattern of collagen in body fluids. The invention further relates to analytical systems to be used when determining the collagen fragmentation pattern. Still further, the invention relates to the use of the above methods to diagnose and characterise the presence of disorders associated with the metabolism of bone.
Diseases of bone, among these osteoporosis, are becoming an increasing burden to society. The total cost in the USA in 1992 of osteoporosis related injuries alone is estimated to be at least USD 10 billion (Riggs, New England Journal of Medicine, 327:620-627 (1992)).
Osteoporosis as well as a number of other diseases of bone are characterised by an increased rate of bone loss when compared to the rate of loss in a healthy population. The rate of loss has been shown to be highly correlated to the future fracture risk (Christiansen et al., Prediction of future fracture risk. In: Christiansen et al, eds, Proceedings 1993. Fourth International Symposium on Osteoporosis, Hong Kong. Osteopress Aps 1993; pp. 52-54). Therefore the rate of loss is an important parameter to estimate for the diagnosis of such diseases.
In order to assess the rate of loss the estimation of the rate of bone resorption plays a key role. Even though the rate of loss is the net difference between the bone formation and bone resorption rates, markers of the bone resorption alone have proved to be good estimates of the rate of loss (Bonde et al. xe2x80x9cImmunoassay for Quantifying Type 1 Collagen Degradation Products in Urine Evaluatedxe2x80x9d Clin. Chem. 40/11, 2022-2025 (1994)xe2x80x94Endocrinology and Metabolism. The estimate of bone loss is improved, however, by including also markers of bone formation (Qvist et al. American Society of Bone and Mineral Research, Abstract # B 419, Kansas City, 1994)
In the past, assays have been developed for monitoring degradation of collagen in vivo by measuring various bio-chemical markers, some of which have been degradation products of collagen.
For example, hydroxyproline, an amino acid largely restricted to collagen, and the principal structural protein in bone and all other connective tissues, is excreted in urine. Its excretion rate is known to be increased in certain conditions, notably Paget""s disease, a metabolic bone disorder in which bone turnover is greatly increased, as discussed further below.
For this reason, urinary hydroxyproline has been used extensively as an amino acid marker for collagen degradation; Singer, F. R. et al., Metabolic Bone Disease, Vol. II (eds. Avioli, L. V., and Kane, S. M.), 489-575 (1978), Academic Press, New York.
U.S. Pat. No. 3,600,132 discloses a process for the determination of hydroxyproline in body fluids such as serum, urine, lumbar fluid and other intercellular fluids in order to monitor deviations in collagen metabolism. The Patent states that hydroxyproline correlates with increased collagen anabolism or catabolism associated with pathological conditions such as Paget""s disease, Marfan""s syndrome, osteogenesis imperfecta, neoplastic growth in collagen tissues and in various forms of dwarfism.
Bone resorption associated with Paget""s disease has also been monitored by measuring small peptides containing hydroxy-proline, which are excreted in the urine following degradation of bone collagen; Russell et al., Metab. Bone Dis. and Rel. Res. 4 and 5, 2250262 (1981), and Singer, F. R., et al., supra.
In the case of Paget""s disease, the increased urinary hydroxyproline probably comes largely from bone degradation; hydroxyproline, however, generally cannot be used as a specific index for bone degradation. Much of the hydroxyproline in urine may come from new collagen synthesis (considerable amounts of the newly made protein are degraded and excreted without ever becoming incorporated into tissue fabric), and from turnover of certain blood proteins as well as other proteins that contain hydroxyproline.
Furthermore, about 80% of the free hydroxyproline derived from protein degradation is metabolised in the liver and never appears in the urine. Kiviriko, K. I., Int. Rev. Connect. Tissue Res. 5:93 (1970), and Weiss, P. H. and Klein, L., J. Clin. Invest. 48:1 (1969). Hydroxyproline is a good marker for osteoporosis as it is specific for collagen in bones even if it is not specific for bone resorption, but it is trouble-some to handle.
Hydroxylysine and its glycoside derivatives, both peculiar to collagenous proteins, have been considered to be more accurate than hydroxyproline as markers of collagen degradation. However, for the same reasons described above for hydroxyproline, hydroxylysine and its glycosides are probably equally non-specific markers of bone resorption; Krane, S. M. and Simon, L. S., Develop. Biochem. 22:185 (1981).
Other researchers have measured the cross-linking compound 3-hydroxypyridinium in urine as an index of collagen degradation in joint diseases. See, for background and as examples, Wu and Eyre, Biochemistry, 23:1850 (1984); Black et al., Annals of Rheumatic Diseases, 45:969-973 (1986); and Seibel et al., The Journal of Rheumatology, 16:964 (1989). In contrast to the present invention, these prior researchers have hydrolyzed peptides from body fluids and then looked for the presence of free 3-hydroxypyridinium residues.
Assays for determination of the degradation of type I, II, and III collagen are disclosed in EP-0394296 and U.S. Pat. Nos. 4,973,666 and U.S. Pat. No. 5,140,103. However, these Patents are restricted to collagen fragments containing the cross-linker 3-hydroxypyridinium. Furthermore, the above mentioned assays require tedious and complicated purifications from urine of collagen fragments containing 3-hydroxypyridinium to be used for the production of antibodies and for antigens in the assays.
At present very few clinical data using the approach described in U.S. Pat. No. 4,973,666 and U.S. Pat. No. 5,140,103 are available. Particularly, no data concerning the correlation between the urinary concentration (as determined by methods described in the above mentioned patents) of 3-hydroxypyridinium containing telopeptides of type I collagen and the actual bone loss (as determined by repeated measurements by bone densiometry) have been published. The presence of 3-hydroxypyridinium containing telopeptides in urine requires the proper formation in bone tissue of this specific cross-linking structure at various times before the bone resorbing process. Very little information on these processes is available and it would be desirable to avoid this dependence on the correct formation of the cross-linking structure.
GE Patent Application No. 2205643 reports that the degradation of type III collagen in the body can be quantitatively determined by measuring the concentration of an N-terminal telopeptide from type III collagen in a body fluid. This method uses antibodies generated to N-terminal telopeptides released by bacterial collagenase degradation of type III collagen, said telopeptides being labelled and used in the assay.
Schr{overscore (o)}ter-Kermani et al., Immunol. Invest. 19:475-491 (1990) describe immunological measurement systems based on CNBr fragments of collagen type I and II. Use is made of pepsin-solubilised collagen, leaving the telopeptides in the tissue (cf. the above mentioned GB Patent Application No. 2205643).
The development of a monoclonal antibody raised against pepsin-solubilised type I collagen is described in Werkmeister et al., Eur. J. Biochem. 1987:439-443 (1990). The antibody is used for immunohistochemical staining of tissue segments and for measuring the collagen content in cell cultures. The measurements are not carried out on body fluids.
EP Patent Application No. 0505210 describes the development of antibody reagents by immunisation with purified cross-linked C-terminal telopeptides from type I collagen. The immunogen is prepared by solubilising human bone collagen with bacterial collagenase. The antibodies thus prepared are able to react with both cross-linked and non-cross-linked telopeptides, and cross-linkers other than pyridinoline.
International Patent Application No. WO 91/09114 discloses certain synthetic peptides which are used to promote cellular adhesion to a solid substrate. The use of the synthetic peptides as immunological reagents is not mentioned.
There are a number of reports indicating that collagen degradation can be measured by quantitating certain collagen propeptides. Propeptides are distinguished from telopeptides and the alpha-helical region of the collagen core by their location in the procollagen molecule and the timing of their cleavage in vivo; see U.S. Pat. Nos. 4,504,587; 4,312,853; Pierard et al., Analytical Biochemistry 141:127-136 (1984); Niemela, Clin. Chem. 31/8:1301-1304 (1985); and Rohde et al., European Journal of Clinical Investigation, 9:451-459 (1979).
EP Patent Application No. 0298210 and No. 0339443 both describe immunological determination of procollagen peptide type III and fragments thereof. Further, a method based on the measurement of procollagen is disclosed in EP Patent Application No. 0465104.
The use of synthetic peptides with sequences derived from type IX collagen for the development of immunological reagents is disclosed in PCT Patent Application No. WO90/08195. Likewise the application describes the use of the antibodies thus produced for the determination of type IX collagen fragments in body fluids.
U.S. Pat. No. 4,778,768 relates to a method of determining changes occurring in articular cartilage involving quantifying proteoglycan monomers or antigenic fragments thereof in a synovial fluid sample.
Dodge, J. Clin Invest 83:647-661 (1981) discloses methods for analysing type II collagen degradation uzilising a polyclonal antiserum that specifically reacts with unwound alpha-chains and cyanogen bromide-derived peptides of human and bovine type II collagens. The degradation products of collagen were not detected in a body fluid, but histochemically by staining of cell cultures, i.e. by xe2x80x9cin situxe2x80x9d detection.
WO94/03813 describes a competitive immunoassay for detecting collagen or collagen fragments in a sample wherein a binding partner containing a synthetic linear peptide corresponding to the non-helical C-terminal or N-terminal domain of collagen is incubated with an antibody to the linear synthetic peptide and the sample, and wherein the binding of the antibody to the binding partner is determined.
WO95/08115 relates to assay methods in which collagen fragments in a body fluid are determined by reaction with an antibody which is reactive with a synthetic peptide. The assay may be a competition assay in which the sample and such a peptide compete for an antibody, possibly a polyclonal antibody raised against fragments of collagen obtained by collagenase degradation of collagen. Alternatively, it may be an assay in which an antibody, possibly a monoclonal antibody, is used which has been raised against such a synthetic peptide.
One particular peptide fragment which we have found in body fluid, particularly urine, is of the formula: 
In the above formula, K-K-K represents cross-link which may for instance be a hydroxypyridinium cross-link but may be any naturally occurring cross-link and specifically any of those discussed in Last et al. Int. J. Biochem. Vol. 22, No. 6, 559-564, 1990.
A larger peptide fragment including the above smaller fragment is reported in EP 0394296.
In one bone resorption assay (CrossLaps(trademark)) described in WO95/08115, fragments of type I collagen containing a specific 8 amino acid sequence of the C telopeptide of type 1 collagen are quantitated (see also Bonde et al., Immunoassay for quanti-fying type I collagen degradation products in urine evaluated, Clin. Chem. 40/11, 2022-2025 (1994)xe2x80x94Endocrinology and Metabolism.
Another bone resorption assay (described in WO 94/03813) relates to all fragments containing a pyridinoline structure and two peptide chains of the N-telopeptides of type I collagen (see also Hanson et al., xe2x80x9cA specific immunoassay for monitoring human bone resorption: Quantitation of type I collagen cross-linked N-telopeptides in urine,xe2x80x9d Journal of Bone and Mineral Research, Vol. 7, Number 11, 1992). We believe the fragments reactive in both these assays to have a considerable variation with respect to their size and their content of crosslinking materials (e.g. pyridinoline, Ehrlich chromogen, and pyrrole structures).
Various studies have been done comparing the results obtained using one prior art assay with those of another such assay on the same samples. The purpose of these studies has been to establish the reliability of these assays as measures of the rate of bone resorption, see for instance Garnero et al, Journal of Clinical Endocrinology and Metabolism, 70, No.3, 780-785.
Whilst studies of this type look for significance in the similarities of the results given by different assays, we believe that they fail to appreciate the valuable information regarding the origin and causes of bone resorption in individual patients which can be revealed by the differences in these results.
The exact fragmentation pattern of type I collagen in vivo is not yet fully elucidated. It has been shown, however, that the fragmentation pattern of type I collagen as measured by the pattern of reactivity in gelfiltration techniques is significantly different in women receiving one anti-resorptive therapy when compared to women receiving another (Garnero et al. American Society of Bone and Mineral Research, Abstract 134, Kansas City, 1994). It has also been shown that the fragmentation pattern varies significantly in untreated women (unpublished observations).
We have now further established that these differences in fragmentation patterns are reflected in differences in results obtained using different immunological assays for bone collagen degradation products.
The present invention now provides a method of estimating the fragmentation pattern of collagen, preferably type 1 collagen, in a body fluid, comprising subjecting a sample of said body fluid to at least two distinct immunological assays, each of which measures the amount of a respective population of collagen breakdown products in said sample and comparing the results of the said measurements.
Thus, in a diagnosis situation, one may aim at measurements of the fragments of type I collagen which put more emphasis on those which may be of xe2x80x9cpathologicalxe2x80x9d nature and put less emphasis on the fragments generated in a xe2x80x9chealthyxe2x80x9d renewal of the skeleton.
It will be understood that the population of breakdown products detected by the respective assays may overlap. Indeed, one of said populations may be a sub-population which is wholly within the other said population.
The use of a comparison, e.g. by forming a ratio, between the concentration of specific fragments and the concentration of other fragments or the sum of fragments is highly relevant in this context as a high rate of bone resorption can probably occur in a xe2x80x9chealthyxe2x80x9d renewal of the skeleton, provided of course that the rate of bone formation also is high. As an example one assay could be used for measurement of the sum of degradation products whereas another would preferentially detect molecules generated during xe2x80x9cnormalxe2x80x9d or xe2x80x9cregularxe2x80x9d collagen degradation. By creating the index between the two assays one will indirectly have information about the amount of collagen fragments generated by xe2x80x9cpathologicalxe2x80x9d collagen degradation, e.g. by bone metastasis. A parallel diagnostic relation exists in the area of estimating the risk of atherosclerosis. In this case the total cholesterol and subfractions of cholesterol in form of HDL and LDL are measured.
According to the invention, the results may preferably be compared by combining them mathematically to form a numerical index, e.g. by taking their ratio.
A ratio formed between the concentration of the fragments measured in two independent immunological assays of bone resorption, provides an index which is dependent on the fragmentation pattern of type I collagen and which therefore can be used for diagnostic purposes in relation to disorders associated to the metabolism of collagen.
A numerical index derived from two or more assays may be linked to a particular identified pattern of fragmentation if desired by separating collagen fragments in the sample, e.g. by HPLC or by gel-filtration, and measuring the amounts of peptide in specific fractions, optionally identifying the peptides in question. However, this is not necessary to the practice of the invention.
One may simply associate particular numerical index results with particular patient types. This may be done by subjecting a range of samples of known type to selected pairs or larger multiples of assays and building up a data-base of results. One may then identify the fragmentation pattern of an unknown sample as being typical of a particular class of sample previously tested. The term xe2x80x9cpatient typexe2x80x9d embraces both healthy patients of different age an/or sex and patients with one or more pathological or abnormal conditions.
Preferably in accordance with the invention each of said populations of breakdown products comprises breakdown products of telopeptides of type I collagen.
Preferably at least one said population is of breakdown products containing peptides comprising one or more of the following amino acid sequences of human type 1 collagen:
Asp-Glu-Lys-Ser-Thr-Gly-Glyxe2x80x94SEQ. ID. No.2
Glu-Lys-Ala-His-Asp-Gly-Gly-Argxe2x80x94SEQ. ID. No.3
Gln-Tyr-Asp-Gly-Lys-Gly-Val-Glyxe2x80x94SEQ. ID. No.4
Gly-Met-Lys-Gly-His-Argxe2x80x94SEQ. ID. No.5
Gly-Ile-Lys-Gly-His-Argxe2x80x94SEQ. ID. No.6
Gly-Phe-Lys-Gly-Ile-Argxe2x80x94SEQ. ID. No.7
Gly-Leu-Pro-Gly-Leu-Lys-Gly-His-Asnxe2x80x94SEQ. ID. No.8
One such population may contain peptides comprising one or more of the following amino acid sequences of human type II collagen:
Glu-Lys-Gly-Pro-Aspxe2x80x94SEQ. ID. No.9
Gly-Val-Lysxe2x80x94SEQ. ID. No.10
Pro-Gly-Val-Lys-Glyxe2x80x94SEQ. ID. No.11
Pro-Gly-Pro-Lys-Gly-Gluxe2x80x94SEQ. ID. No.12
Gly-Gln-Lys-Gly-Glu-Proxe2x80x94SEQ. ID. No.13
or
Gly-Asp-Ile-Lys-Asp-Ile-Valxe2x80x94SEQ. ID. No. 14
or one or more of the following amino acid sequences of human collagen type III:
Asp-Val-Lys-Ser-Gly-Valxe2x80x94SEQ. ID. No. 15
Glu-Lys-Ala-Gly-Gly-Phe-Alaxe2x80x94SEQ. ID. No. 16
Gly-Phe-Pro-Gly-Met-Lys-Gly-His-Argxe2x80x94SEQ. ID. No. 17
or
Gly-Ala-Ala-Gly-Ile-Lys-Gly-His-Argxe2x80x94SEQ. ID. No. 18
Similar sequences with isoaspartic acid replacing aspartic acid may be detected.
Preferably, one of said assays measures the amount of population of breakdown products characterised by containing isoaspartic acid.
Said population may comprise or consist o breakdown products containing one or more peroxides of the sequence EKAH*GGR, wherein * is isoaspartic acid and K is part of a collagen cross-link or is lysine.
One of the assays may involve determining the amount of the peptide of formula 2 (below) present in said body fluid: 
wherein K-K-K is any naturally occurring cross-link and * is isoaspartic acid, or of one or more peptides incorporating an epitope present in the peptide of formula 2 which contains isoaspartic acid.
Said determination may be carried out using an immunological binding partner specific for an isoaspartic acid containing species present in the sample during the procedure.
The immunological binding partner may be an antibody raised against a linear peptide corresponding to a sequence within collagen with isoaspartic acid substituting in said amino acid sequence for aspartic acid in said collagen sequence. It may be an antibody raised against a fragment of collagen, selected for its affinity for such an isoaspartic acid containing peptide.
A or the other said assay may preferably measure a population of breakdown products containing peptides related to those detected in said one assay by the presence of aspartic acid in place of isoaspartic acid.
The invention includes a kit for use in estimating the fragmentation pattern of collagen type 1 in a body fluid, comprising an immunological binding partner for a first population of collagen type I breakdown products, an immunological binding partner for a second population of collagen type 1 breakdown products and optionally one or more assay kit ingredients selected from buffers, wash solution, synthetic peptides, anti-idiotype antibodies, antibody-enzyme conjugates, substrates for antibody-enzyme conjugates, body fluid control samples, standard solutions and enzyme conjugate reaction stopping solutions.
In accordance with a particularly preferred practice of the invention, we have found that specific fragmentation patterns of type I collagen, as determined in urine samples of gelfiltration techniques, can be estimated by forming a ratio between results obtained using two immunological assays, both assays being assays of bone resorption and both measuring degradation products of type I collagen. A first of the assays is based on a polyclonal antibody and is described in Bonde et al., Immunoassay for quantifying type I collagen degradation products in urine evaluated, Clin. Chem. 40/11, 2022-2025 (1994)xe2x80x94Endocrinology and Metabolism. The second assay is based on a monoclonal antibody and is described in Fledelius et al., American Society of Bone and Mineral Research, Abstract C 344, Kansas City, 1994). Both of these assays are also described in WO 95/08115.
It is observed, using gelfiltration experiments on urine samples, that the degradation of type I collagen varies from individual to individual not only in a quantitative manner but also in a qualitative manner. In order to express the qualitative differences in the degradation of type I collagen in a quantitative manner, a ratio may be formed between the results obtained in each of the above mentioned assays detecting different degradation fragment of type I collagen.
This ratio can be used for distinguishing between urine samples giving identical readings in one or other assay (see Table 1), and therefore has utility for diagnostic purposes.
It is contemplated that the method of forming the relevant ratio between assays of bone resorption will be used to diagnose disorders of the metabolism of collagen analogously to the diagnosis and estimation of risk of atherosclerosis, namely by measuring the total cholesterol and subfractions (HDL, LDL) and by forming the relevant ratios between the subfractions and the total cholesterol.
In brief the assays referred to above are based on an immobilised synthetic peptide with an amino acid sequence found in a part of the C-terminal telopeptide of the xcex1I chain of type I collagen (Glu-Lys-Ala-His-Asp-Gly-Gly-Arg=8AA).
To produce the polyclonal antibody used in the first assay, rabbits were immunised with collagenase treated collagen and antibody serums reactive with 8AA were selected.
To produce the monoclonal antibody of the second assay rabbits were immunised with 8AA conjugated to BSA using a two step carbodimide procedure.
For coating of microtitre plates and 8AA peptide was conjugated to thyroglobulin using glutaraldehyde (Soinila S, Mpitsos G J, Soinila J. Immunohistochemistry of encephalins: Model studies on hapten-carrier conjugates and fixation methods. J. Hitochem Cytochem 1992:2:231-9). During incubation of samples with these antibodies a competition takes place between the immobilised peptide and the breakdown products of type I collagen in urine. As the content of the peptide in the solution increases, less antibody will bind to the immobilised peptide leading to a decreasing optical density.
Surprisingly it has been found that whilst the monoclonal antibody successfully detects peptides in urine containing all or some of the 8AA sequence, the polyclonal antibody under assay conditions selectively detects in urine peptides containing all or some of an analogous amino acid sequence in which isoaspartic acid replaces aspartic acid in the 8AA sequence (iso-8AA). In place of such a polyclonal antibody one may therefore use a polyclonal antibody selected for reactivity with the iso-8AA peptide or a monoclonal antibody raised against iso-8AA.
Thus, we have now discovered that a proportion of the peptide fragments in body fluids are related to peptides of equivalent formula, e.g. pertides of formula 1, by their replacement of aspartic acid in the formula by isoaspartic acid.
The isomerization of aspartic acid has been reported previously to be a spontaneous reaction occurring under physiological conditions.
See for instance Brennan et al Protein Science 1993, 2, 331-338, Galletti et al, Biochem. J. 1995, 306, 313-325, Lowenson et al, Blood Cells 1988, 14, 103-117 and Oliya et al, Pharmaceutical Research, Vol. 11, No. 5, 1994, p.751.
The above discovery indicates that this isomerization also occurs in bone tissue and the extent of isomerization is expected therefore to be marker for the age of the bone tissue concerned.
Furthermore, the presence in such bone peptide fragments of the isomerization provides confirmation that the peptide fragments indeed derive from bone degradation and not some other source such as the degradation of newly formed collagen never incorporated into bone.
Preferably, therefore one of the assays is carried out using an immunological binding partner specific for an isoaspartic acid containing species present in the sample during the procedure, preferably said peptide of formula 2 or a peptide incorporating an epitope present in the peptide of formula 2 which contains isoaspartic acid.
The immunological binding partner may be a monoclonal or polyclonal antibody. By the requirement that the immunological binding partner be specific for the isoaspartic acid containing species is meant that the immunological binding partner distinguishes between said species and the analogous aspartic acid containing species to an extent useful in the assay.
Suitable immunological binding partners also include Fragments of antibodies capable of binding the same antigenic determinant including Fab, Fabxe2x80x2 and F(abxe2x80x2)2. fragments.
Preferably, the immunological binding partner is an antibody raised against a linear peptide, preferably a synthetic peptide, corresponding to a sequence within collagen with isoaspartic acid substituting in said amino acid sequence for aspartic acid in said collagen sequence
Each assay may take many forms including ELISA, RIA, or IRMA, procedures for which are too well known to warrant description here.
In an ELISA of this type, the synthetic peptide may be immobilised on a solid support. A sample may be incubated with a polyclonal antibody or monoclonal antibody for the synthetic peptide in contact with the solid support and after washing, a peroxidase-conjugated (revealing) antibody may be added. After further incubation, a peroxidase substrate solution is added. By competition, peptides in the sample reactive with the antibody inhibit the peroxidase reaction.
Where the synthetic peptide is used to raise a monoclonal immunological binding partner, the synthetic peptide need not be a competing agent in the assay. For instance, collagenase treated collagen may be purified and immobilised onto the solid support and an ELISA may be carried out using a monoclonal antibody.
Antibodies may be prepared which are respectively selective for one or more aspartic acid containing peptides and for their isoaspartic acid containing analogues. It is then possible to carry out an assay for both variants of the peptide or peptides. The relative amount of isoaspartic acid will provide an indication of the age of the bone which is being broken down. Accordingly, the invention provides a method of obtaining information regarding collagen resorption in a patient, comprising measuring in a body fluid the relative amounts of at least one aspartic acid containing peptide derived from collagen and a corresponding isoaspartic acid containing peptide.
The invention may be include both to humans and to animals.
Suitable body fluids include, e.g. human, urine, blood, serum, plasma and synovial fluid. It is contemplated that the method may also be used e.g. on saliva and sweat. The body fluid may be used as it is, or it may be purified prior to the contacting step. This purification step may be accomplished using a number of standard procedures, including, but not limited to, cartridge adsorption and elution, molecular sieve chromatography, dialysis, ion exchange, alumina chromatography, hydroxyapatite chromatography, and combinations thereof.